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Simultaneous Quantitation of Multiple Classes of Organohalogen Compounds in Fish Oils with Direct Sample Introduction Comprehensive Two-Dimensional Gas Chromatography and Time-of-Flight Mass Spectrometry EUNHA HOH,*,†,‡ STEVEN J. LEHOTAY,*,† KRISTIN C. PANGALLO,§,| KATERINA MASTOVSKA,† HELEN L. NGO,† CHRISTOPHER M. REDDY,| AND WALTER VETTER⊥ Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, MIT/WHOI Joint Program in Oceanography/Applied Ocean Science and Engineering and Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, and Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, D-70599 Stuttgart, Germany
We successfully optimized an analytical method using gel permeation chromatography followed by direct sample introduction comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry to quantify multiple groups of targeted persistent organic pollutants and halogenated natural products (HNPs) simultaneously in fish oil samples. This new method has a wider analytical scope than the traditional approach to use multiple methods to cover each class of compounds. Our analysis revealed that the relatively more volatile and lighter organic compounds, such as polychlorinated biphenyls (PCBs), organochlorine pesticides, and other smaller organohalogen compounds, were still present in two brands of “PCB-free” cod liver oils, albeit at much lower levels than in an untreated commercial sample. Moreover, the less volatile organic compounds, such as polybrominated diphenyl ethers and brominated HNPs, were detected at similar levels in all three cod liver oils. This suggests that the commercial molecular distillation treatment used for removal of organic/inorganic toxic contaminants is only effective for the lighter organic contaminants. KEYWORDS: Direct sample introduction; comprehensive two-dimensional gas chromatography; timeof-flight mass spectrometry; persistent organic pollutants; halogenated natural products; dietary supplements; fish oil
INTRODUCTION
Anthropogenic persistent organic pollutants (POPs), such as polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and polybrominated diphenyl ethers (PBDEs), are ubiquitous in the environment. They are persistent and bioaccumulative and have thus entered both terrestrial and marine food webs. Human exposure to PCDD/Fs and PCBs is mainly * To whom correspondence should be addressed. E-mail: (E.H.)
[email protected] and (S.J.L.)
[email protected]. † U.S. Department of Agriculture. ‡ Current Address: Graduate School of Public Health, San Diego State University, San Diego, CA 92182. § MIT/WHOI Joint Program in Oceanography/Applied Ocean Science and Engineering. | Woods Hole Oceanographic Institution. ⊥ University of Hohenheim.
through food; specifically, the consumption of fish is a major exposure pathway for these toxic pollutants in humans (1, 2). In most cases, human and wildlife exposure studies have been limited to the POPs (PCDD/Fs, PCBs, OCPs, and PBDEs), and specific analytical methods have been devised for their analysis. Recently, multiple groups have reported the presence of lipophilic and bioaccumulative halogenated natural products (HNPs) in marine organisms (3-6) (see Figure 1 for HNPs chemical structures). In addition, Pangallo and Reddy showed the possibility of biomagnification of halogenated 1′-methyl1,2′-bipyrroles (MBPs) (4). However, most studies cover a single or a few groups of POPs or HNPs using a targeted analytical approach, which includes laborious sample preparation followed by multiple injections in gas chromatography-mass spectrometry (GC/MS) (7-10). A better risk analysis of POPs to humans and the environment requires data on multiple groups of POPs in several matrices. Hence, the development and usage of a
10.1021/jf900462p CCC: $40.75 2009 American Chemical Society Published on Web 03/05/2009
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Figure 1. Chemical structures of PBDEs, PCBs, and HNPs.
single analytical method would provide the most efficient and cost-effective way to collect such data. In a previous study, we reported the qualitative capabilities of an analytical approach using direct sample introduction comprehensive two-dimensional gas chromatography with timeof-flight mass spectrometry (DSI-GC×GC/TOF-MS) and gel permeation chromatography (GPC) cleanup, which allowed identification of targeted POPs as well as untargeted organic chemicals in fish oil samples (11). This combined analytical approach efficiently increases the scope of organic chemicals monitored in the fish oils: GPC cleanup removes the bulk of the oil matrix, DSI enables large volume injection in a rugged manner, GC×GC provides greater separation and sensitivity than GC alone, and TOF-MS collects full mass spectra. The greater selectivity of GC×GC affords cleaner mass spectra in complex extracts (fewer interferences), which helps in quantitation and qualitative identifications of targeted and nontargeted chemicals. Using DSI-GC×GC/TOF-MS, we found anthropogenic POPs (PCBs, OCPs, and PBDEs) in a dietary supplement of cod liver oil that was labeled “PCB\heavy metal free” (11). In the sample, we also identified and confirmed several groups of HNPs [MBPs, 1,1′-dimethyl-2,2′-bipyrroles (DMBPs), methoxylated PBDEs (MeO-PBDEs), polybrominated hexahydroxanthene derivatives (PBHDs), polybromoindoles, and a halogenated monoterpene MHC-1] in addition to other organic contaminants (oxybenzone and octachlorostyrene) (11). In this study, our objectives were to demonstrate the feasibility and applicability of the new analytical approach in a simultaneous and robust quantification of POPs, HNPs, and other organic contaminants in commercially available dietary fish oil supplements with or without treatment for PCB removal. EXPERIMENTAL PROCEDURES Samples. Alaskan sockeye salmon fish oil supplement in capsules was purchased from an Internet retailer. Two dietary cod liver oil supplements (liquid oil) were also purchased from an Internet retailer; one bottle had a label indicating “PCB/heavy metal free”, and the other did not. A third bottle of dietary cod liver oil supplement (liquid oil) was obtained from a local supermarket in Maryland; the bottle label indicated that the cod liver oil was molecularly distilled, and heavy metals, PCDDs, and PCBs were undetected.
Materials. PCB standard mixtures (PCB-W22), OCP mixture (M-680P), PBDE standard mixture (BDE-CM, PBDE congeners of primary interest calibration mix), individual PCB standards (CB65, 155, and 204), individual PBDE standards (BDE77 and 166), octachlorostyrene, and two MeO-PBDE mixtures (2′-MeO-BDE68 and 6-MeOBDE47) were all purchased from AccuStandard (New Haven, CT). 13 C12-Labeled PCB standard mixture (CB 77, 81, 126, and 169) and 13 C12-CB189 were purchased from Wellington Laboratories (Guelph, Canada). 5H-Bromoindole, 2,4,6-tribromoanisole, oxybenzone, and 1,4dimethoxy-2,5-dimethoxybenzene were purchased individually from Sigma-Aldrich (Milwaukee, WI), and 4,6-dibromoindole was purchased from SiNova (Bethesda, MD). Many HNPs, for which reference standards were not commercially available, were made in laboratories. The molecular structures and their acronyms are shown in Figure 1, and their purities and sources are as follows: Standards of halogenated MBPs (MBP-HBr5Cl, MBP-HBr6, MBP-HBr6Cl, and MBP-Br7) were extracted from the blubber of a common dolphin and isolated by GPC and silica column followed by preparatory capillary GC (purity >95%) (12). Q1 (MBP-Cl7) was synthesized and recrystallized (purity >98%) (13); MHC-1 (purity >90%) was calibrated with trans-chlordane (14); and two congeners of PBHDs were isolated from Mediterranean sponge (purity >95%) (15). 2,2′-Dimethoxy-3,3′,5,5′-tetrabromobiphenyl (2,2′-diMeO-BB 80) was synthesized following the procedure described by March et al. (16) (purity >95%). Standards of halogenated DMBPs (two isomers of DMBP-Br3Cl3, DMBP-Br4Cl2, DMBP-Br5Cl, and DMBP-Br6, purity >99%) were donated by Sheryl Tittlemier (Health Canada, Ontario, Canada). All solvents used in this study were high-performance liquid chromatography grade; cyclohexane and toluene were purchased from Sigma-Aldrich, and ethyl acetate (EtOAc) and isooctane were purchased from J. T. Baker (Phillipsburg, NJ). Septa for the autosampler vial caps were heated at 200 °C overnight, and all glassware and glass wool were heated at 450 °C for 6 h in a muffle furnace prior to use. Sample Preparation. For lipid removal from cod liver oil extracts, an automated GPC instrument (J2 Scientific, Columbia, MO) was employed. The glass GPC column was 2 cm i.d. and 22.5 cm length packed with 24 g of BioBeads S-X3 in 1:1 EtOAc:cyclohexane (v:v) and was purchased from J2 Scientific. Fish oil (0.5 g) was dissolved into the mobile phase 1:1 cyclohexane:EtOAc solvent with spiked known amounts of internal standards (CB65, 155, and 204 and BDE77 and 166) and brought to 10 mL of volume. Half (5 mL ) 0.25 g) of each sample was injected into the GPC at 5 mL/min, and elution was between 12.5 and 22.5 min and reduced finally to 100 µL with final
Organohalogen Compounds in Fish Oils addition of recovery standards (13C12-labeled CB77, 81, 126, 169, and 189) in isooctane. For more sensitive analysis of heavier HNPs, a larger sample size of 1 g instead of 0.25 g was used but with two GPC cleanup steps. Instrumental Parameters. A Pegasus 4D (Leco, St. Joseph, MI) GC×GC/TOF-MS was used in this study with the following column configuration: a Restek (Bellefonte, PA) Siltek deactivated column (5 m, 0.25 mm i.d.) as a guard column, a Restek Rtx-5Sil-MS (15 m, 0.25 mm i.d., 0.25 µm film thickness) as the 1D column, and a J&W Scientific (Folsom, CA) DB-17MS (2 m, 0.18 mm, 0.18 µm thickness) as the 2D column. Ultrapurity He (Airgas, Radnor, PA) was used as the carrier gas. The instrument conditions were as follows: The primary oven temperature was set at 60 °C for 7.5 min, ramped at 10 °C/min to 300 °C, and held for 20 min; the secondary oven temperature was programmed to be 20 °C higher than the primary oven; for GC×GC, the modulation period was set as 3.5 s with 0.9 s hot pulse duration and 35 °C modulator temperature offset vs the primary oven temperature; MS transfer line, 270 °C; ion source temperature, 250 °C; electron energy, -80 eV; detector voltage, 1850 V; and data acquisition rate, 100 spectra/s for the m/z range of 50-800. Injection was conducted by a Combi-PAL autosampler (Leap Technologies; Carrboro, NC) with the automated DSI accessory (Linex) in combination with an Optic 3 programmable temperature vaporizer (Atas-GL International; Veldhoven, The Netherlands). The injection volume was 10 µL. The optimized conditions for DSI of final extracts in isooctane were as follows: initial injector temperature at 70 °C for 8 min with 50:1 split ratio (to vent the evaporated solvent), ramped to 320 °C at the maximum rate (12 °C/s) with a splitless period of 7.5 min, then 50:1 split ratio for 16.5 min, at which point the split flow was reduced to 25:1 and the injector temperature was cooled to 250 °C. The carrier gas flow rate was held at 1 mL/min for 8 min, ramped to 2.5 mL/min as a pressure pulse during the 7.5 min splitless period, then reduced to 1 mL/min until 33 min, and ramped to 1.5 mL/min until the end of the analysis. Data Processing and Quality Control. Data analysis was conducted with the Leco ChromaTOF (version 3.25) software. Quantitation of the analytes for which reference standards were available used five levels of calibration standards. Manual review was made of all integrations and identifications to ensure accuracy of the results. The average recovery of the six internal standards was 91% with an overall range of 64-122% among the sample analyses and quality control spikes. The PCB analysis was not fully congener-specific, which is a very complicated undertaking due to coelutions of PCB congeners (17). Instead, to simplify the analysis without affecting overall PCB quantitation, we used 15 PCB congeners including seven PCB indicators (CB28, 52, 101, 118, 138, 153, and 180). Some PBDEs, oxybenzone, and a dibromoindole were also detected in the “blank” samples, so only those results more than three times higher than the blank levels are reported. A few other analytes were detected in the blank samples but at such low levels that there was no need to correct for their concentrations in the samples. RESULTS AND DISCUSSION
Method Optimization for Analysis of PCBs, OCPs, and PBDEs in Fish Oil. The analytical approach using DSIGC×GC/TOF-MS only requires GPC cleanup for fish oils based on the previous sample cleanup comparison study (11). Fankhauser-Noti and Grob developed a GC procedure using backflushing in the inlet for injection of diluted edible oils (18), but their approach was not demonstrated to be effective for relatively nonvolatile components such as PBDEs. The equivalent amount of sample injected in their method was 200 µg, which is too little to achieve very low detection limits for organic contaminants. Through the initial use of GPC in our study, we injected 25 mg of equivalent sample. Without the use of GPC, the presence of too much oil in the removable microvial reduces transfer efficiency of lipophilic analytes into the GC column, and quantitation suffers.
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Figure 2A shows comparison of responses of selected PCBs and PBDEs in cod liver oil of different sample sizes (0.1, 0.25, 0.5, and 1 g) after GPC cleanup by injection using microvials in DSI. The peak responses of the more volatile, smaller compounds increased as the amount of cod liver oil sample increased, as would be expected with increasing analyte concentration in the final extracts. However, the peak responses of the less volatile, larger compounds remained fairly constant regardless of the sample size because more lipids remained in the final extracts as sample size in GPC increased, which caused worse transfer efficiency of the heavier compounds from the DSI microvial to the GC column. Therefore, use of an appropriate amount of injected sample or a larger GPC column for larger samples was an option to better remove the lipids. Because the GPC column was fixed in this study, we chose a smaller sample size to reduce the amount of lipid coextractives and improve transfer efficiency for less volatile analytes in DSI. On the basis of the result in Figure 2A, 0.25 g was chosen as an optimal injected amount of oil. Additionally, a higher GC injection temperature improved the transfer efficiency of the heavier analytes. Also, the use of several internal standards with different molecular weights corrected for the different analyte transfer efficiencies on account of the lipids in the final extracts (see Figure 2B). In a final optimized method, we added a heavier internal standard, hexaBDE (BDE166), to better correct for the lower transfer efficiency of PBDEs with five or more bromines (gBr5). Limits of detection (LODs) calculated based on signal/noise of 3 using the instrument software were 0.44 pg/g for BDE77 and 0.63 pg/g for 13C-CB189. We quantified selected PCBs, PBDEs, and OCPs in three samples from each of the three dietary cod liver oils with the optimized analytical method, and results are summarized in Table 1. The quantified concentrations of all analytes were consistent with a relative standard deviation (RSD) of 11% on average, and 95% of the total data had RSDs between 1.0 and 25%. PCBs, OCPs, PBDEs, Lighter Halogenated Compounds, and Oxybenzone. Table 1 summarizes the concentrations of selected POPs (OCPs, PBDEs, and PCBs including seven indicators: CB28, 52, 101, 118, 138, 153, and 180), the relatively more volatile halogenated compounds, and oxybenzone that were previously confirmed and identified in the cod liver oil, sample 2 (11). The three cod liver oils are named as samples 1, 2, and 3 as indicated in Table 1. Two cod liver oil supplements, samples 1 and 2, were from the same company, and their source was Norwegian cod liver oil, but only sample 2 had a label of PCB/heavy metal free with the written claim, “This product has been processed and tested to be free of PCB and heavy metals”. The third cod liver oil sample was from a different company, but its source was from Arctic cod liver oil (similar to the other two samples), and the bottle was labeled “molecularly distilled” and “undetected levels of heavy metals, dioxins, and PCBs”. When contacted, the vendor of the “PCB/heavy metal free” bottle also stated that molecular distillation was conducted by the supplier in Norway. This process involved cooking and pressing of the cod fish at
